Download Determination of Carbamazepine in Pharmaceutical

Determination of Carbamazepine in Pharmaceutical Preparations and Pure
Forms Using HPLC-DAD, First Order- and Second Order- Derivative
Spectrophotometric Methods
Fatma DEMİRKAYA*, Yücel KADIOĞLU*

*Department of Analytical Chemistry, Faculty of Pharmacy, Ataturk
University, 25240, Erzurum, TURKEY( Tel:+90 442 2311536 ;email:[email protected])
Abstract: HPLC-DAD, First order- and Second order- derivative spectrophotometric methods were
developed and validated for quantitative determination of Carbamazepine (CBZ) in three different
pharmaceutical preparations. Validation parameters such as linearity, precision, accuracy, specificity,
limit of detection (LOD) and limit of quantitation (LOQ) were determined according to International
Conference on Harmonization guidelines. HPLC-DAD was carried out by reversed phase technique on
Bondolone C18 column with a mobile phase composed of acetonitrile and Milli-Q grade water (30:70,
v/v) pumped at a flow-rate of 1 ml min-1 at 220 nm. Spectrophotometrically, CBZ was determined by
measuring the first order- derivative values at 285 nm and second order- derivative values at 287 nm.
Beer’s Law was obeyed in the range 0.25-25 g ml-1 for HPLC and 1.25-25 g ml-1 for first order- and
second order- derivative spectrophotometric methods. Statistical analysis by F-test showed no significant
difference between the results obtained by three methods. The proposed methods are highly sensitive,
precise and accurate and can be used for the reliable quantitation of CBZ in pharmaceutical preparations.
Keywords: Carbamazepine, HPLC, First order- derivative and Second order- derivative

1. INTRODUCTION
Carbamazepine (CBZ), 5-H-Dibenz [b.f] azepine-5carboxomide (Fig.1.) is widely prescribed on an
anticonvulsant, antiepileptic and antimanic drug (Olling et al,
1999). In the body, CBZ is metabolized to an active
metabolite CBZ-10.11 Epoxide (CBZ-EP) which also
displays anticonvulsant properties similar to these of the
parent compound. CBZ is poorly soluble in aqueous media
and has a high oral bioavailability in humans (Jung et al,
1997).
Several Methods have been reported for the determination of
CBZ standarts and in pharmaceutical preparations including
Spectrophotometric Methods (Riad et all, 1986, Camara et
all, 2005, Rezaei et all, 2005), Spectrofluorimetry Methods
(Huang et all, 2002, Fellenberg et all, 1976), Gas-Liquid
Chromatograpy (GC) [Frigerio et all, 1973, Chen et all,
1991), FT-Raman Spectroscopy (Auer et all, 2003), Liquid
Chromatograpy [Mohammed, 2000, Manoj Babu, 2004, Lam
et all, 2004, González-Barreiro et all, 2003). But there isn’t
any Derivative UV Spectrophotometric Methods for the
determination of CBZ in pharmaceutical preparations.
Derivative UV spectroscopy has been widely used as a tool
for quantitative analysis, characterization, and quality control
in agricultural, pharmaceutical, and biomedical fields. This
technique offers various advantages over the conventional
absorbency methods such as the discrimination of the sharp
spectral features over the large bands and the enhancement of
the resolution of overlapping spectra (Wang et all,2000, ElGindy et all, 2000). The aim of this study was to develop a
HPLC
method
and
an
alternative
derivative
Spectrophotometric method, to the more time consuming
HPLC method, which can be used regularly and for
formulation screening. HPLC-DAD, first order- and second
order- derivative spectrophotometric methods that could be
used for determination of CBZ in pure forms and in
pharmaceutical preparations were developed and validated.
The results obtained from these methods were statistically
compared. Statistical analysis (F-test) showed no significant
difference between the results obtained with three methods.
Fig.1.Chemical structure of Carbamazepine.
2. EXPERIMENTAL
2.1. Materials and Chemicals
CBZ was kindly supplied by Novartis (Turkey). HPLC grade
and analytical grade methanol and acetonitrile were
purchased from Merck (Germany). The water of HPLC was
prepared by double glass distillation and filtration through
Millipore 0.45 m. white nylon HNWP 47 mm filters.
Three commercial preparations were used in assay including;
Tegretol tablet (Novartis Pharmaceuticals, Turkey)
containing 200 mg CBZ per tablet, Karberol tablet (Munir
Sahin Pharmaceuticals, Turkey) containing 200 mg CBZ per
tablet and Karbelex tablet (Liba Pharmaceuticals, Turkey)
containing 300 mg CBZ per tablet
2.2. Spectrophotometric Assay
A Thermospectronic (HEIOS) double-beam UVVisible spectrophotometer with a Vision 32 software data
processing system was used. Derivative spectra of the
solutions were recorded in 1 cm quartz cells at a scan speed
of 600nm/min, a scan range of 270-320 nm, fixed slit width
of 2 nm and derivation interval () 21.0 nm.
2.3.Chromatographic Assay
The CBZ analyses were performed on a
Thermoseparation Spectra HPLC system consisting of a UV
6000 LP photodiode array detector, Series P 400 gradient
pump and a Thermoseparations As 3000 autosampler.
Isocratic elution was carried out at a flow-rate of 1 ml min-1
with the mobile phase containing acetonitrile-Milli-Q grade
water (30:70 v/v) pumped through a Phenomenex Bondolone
reversed-phase C18 column (150 x 3.9 mm. 5 m. “USA”)
The injection volume was 10 l and the peaks were detected
at 220 nm. Retention time of CBZ was 8.2 min and the total
run time for an assay was approximately 9 min.
2.4. Preparation of Standard Solutions
Standard stock solution of CBZ was prepared with
methanol to a concentration 50 g ml-1 and stored at 4°C. The
solution was stable for at least 2 month when kept in
refrigerator. The working standard solutions were prepared
by dilution of different volumes of the stock solution to a
constant volume with methanol to reach a concentration
range of 1.25-25 g ml-1. First order- and second orderderivative curves of working standard solutions were scanned
270-320 nm against a similarly prepared blank. Calibration
graphs of first order- and second order- derivative (N=9.
=21.0 nm) spectrophotometric methods was determined by
plotting the absorbances versus CBZ concentration at 285 nm
and 287 nm respectively.
For HPLC-DAD analysis, 0.25-25 g ml-1 working
solutions were also prepared from stock solution and diluted
by mobile phase. Working solutions were prepared freshly in
analysis daily. The solutions were filtered through a
phenomenex membrane of 0.45 m pore size (25 mm filter)
and transferred to an autosampler vial for analysis.
Calibration graphs were prepared by using peak-area versus
concentrations of CBZ in the solutions.
2.5. Analysis of Pharmaceutical Preparations
Twenty tablets of each drug (Tegretol®. Karberol® and
Karbelex® tablets) were weighed and finely powdered. A
portion of the powder, equivalent to about 10 mg CBZ for
each drug, was accurately weighed, transferred into 50 ml
volumetric flask and dispersed in 40 ml methanol. The flask
was placed in ultrasonic bath for 15 min. The resulting
suspension was diluted to volume with methanol and filtered.
Further dilutions were performed to suit the calibration
graphs for the HPLC-DAD, first order- and second orderderivative spectrophotometric methods.
2.6. Method Validation
The methods were validated according to International
Conference on Harmonisation guidelines (Commision of the
European Communities, 1996) for validation of analytical
procedures. All statistical calculations were performed with
the Statistical Product and Service Solutions (SPSS) package
for windows, version 10.0 Correlations were considered
statistically significant if calculated P values were 0.05 or
less.
3. RESULTS AND DISCUSSION
3.1. The First Order- and Second Order- Derivative
Spectrophotometric Methods
To develop a sensitive the first order- and second
order- derivative spectrophotometric method, various solvent
systems were tried, such as water, methanol and acetonitrile
individually or in combinations of different proportions. The
final decision of using methanol was based on sensitivity,
interference, easy of preparation, suitability for drugs, content
estimation and cost in that order.
The first order- derivative spectrum and second orderderivative spectrum of CBZ standard solutions are shown
Fig.2A and 3A, respectively. Spectra with higher order of
derivation had lower sensitivity and linearity, and because of
this, first order- and second order- derivative spectra were
selected for quantitative analysis. The first order- and second
order- derivative spectrum of solutions of pharmaceutical
preparations containing CBZ presence of interfering
compounds in tablet formulations are also given Fig.2B-D
and 3B-D. For the determination of CBZ in pharmaceutical
dosage forms, the peak-to-peak amplitudes in the first-order
and second order- derivative spectra at 285 nm and 287 nm,
respectively, (peak-to-peak amplitude) was measured with a
good sensitivity and linearity. The values of the first-order
and second order- derivative concentration of citied drugs can
be directly calculated since first-order and second order
derivative measurement cancels the irrelevant absorbance due
to the excipients in pharmaceutical dosage forms.
In general, the characteristic profiles of the derivative
spectra may constitute a specific fingerprint useful for the
drug identification the ratios, in particular, between the
amplitudes at selected wavelength can be regarded as suitable
parameters which are useful to confirm the drug identity,
purity and stability.
shown in this figure, CBZ was eluted forming symmetrical
peak and well separated from the solvent front. Observed
retention time (8.2 min) allows a rapid determination of the
drug. System suitability parameters calculated under the
optimized experimental conditions were; capacity factor (k ’)
4.46; symmetry factor 1.05 and column efficiency (n) 4303
plates /m.
Fig.2. Firs order- derivative spectra of 10 g ml-1 CBZ: A)
Standard B) Tegretol C)Karbelex D)Karberol
Fig.4. HPLC chromatogram of 10 g ml-1 CBZ: A)
Standard B) Tegretol C)Karbelex D)Karberol
3.3. Validation of the Proposed Methods
Fig.3. Second order- derivative spectra of 10 g ml-1 CBZ:
A) Standard B) Tegretol C)Karbelex D)Karberol
3.2. HPLC-DAD Method
In order to evaluate the results of the absorption (zero-order)
UV and the first-order derivative spectrophotometric
methods, a HPLC-DAD procedure was developed. The
development of the HPLC-DAD method for the
determination of drugs has received considerable attention in
recent years because of their importance in routine quality
control analysis. A reversed-phase HPLC-DAD method was
proposed as a suitable method for the estimation of CBZ in
pharmaceutical dosage form. The chromatographic
conditions were adjusted in order to provide a good
performance of the assay. The method involved a
Phenomenex Bondolone reversed-phase C18 column and a
mobile phase consisting of acetonitrile- Milli-Q grade water
(30:70 v/v). The mobile phase was chosen after several trials
with other solvent combinations. Mobil phase selection was
based on peak parameters (symmetry, tailing), run time easy
of preparation and cost. Fig.4A-D shows a typical
chromatogram obtained from the analysis of a standard and
solution of commercial CBZ using the proposed method. As
3.3.1 Linearity
To determine the linearity of HPLC-DAD, first
order- and second order- derivative spectrophotometric
methods, calibration standard solutions of CBZ were
prepared as in the text. The linear ranges were found to be
0.25-25 g ml-1 for HPLC and 1.25-25 g ml-1 for first orderand second order- derivative spectrophotometric methods.
Linearity was estabilished by least-squares regression
analysis. The correlation coefficients, r, for HPLC-DAD, first
order- and second order- derivative spectrophotometric
methods were 0.9995, 0.9840 and 0.9850 (n=6) (Table 1).
Table 1: Results of regression analysis of data for the
quantitation of CBZ by the proposed methods (n=6)
: wawelength, aBased on six calibration curves, LR: linear regression, Sa:
Standard deviation of intercept of regression line Sb: Standard deviation of
slope of regression line, R: Coefficient of corelation, y: Absorbans, y1: Peakarea,x: Carbamazepine concentration (g ml-1)
3.3.2.Stability
The stability of CBZ was determined with HPLC
method. For the determination of the stability of CBZ in
solution at ambient 4oC and -20oC refrigeration temperature,
three sets of low (2.5 g ml-1 ), medium (7.5 g ml-1) and
high (15 g ml-1) concentrations were divided into 12 tubes.
One set was assayed immediately and taken as standard (100
%). One of the sets was stored at a temperature of 4 oC for 24
h and 1 week. Other set was stored at a temperature – 20oC in
a deepfeezer for 24 h. The remaining set was allowed to stand
at ambient temperature for 24 h. Then the stability
measurements were carried out. The results were evaluated
comparing these measurements with those of standards and
expressed as percentage deviation. They are summarized in
Table 2.
LOD and LOQ were determined by an empirical method that
consisted of analyzing a series of standard solutions
containing decreasied amounts of CBZ. The limit of
quantification (LOQ) is defined as the lowest concentration
on the calibration curve at which both accuracy and precision
should be within %20. LOQ and LOD values were
determined to be 0.07 g ml-1 and 0,05 g ml-1 for HPLCDAD and 0.50 g ml-1and 0,25 g ml-1 for first order- and
second order- derivative spectrofotometric methods,
respectively, whose precision and accuracy were well within
the proposed criteria.
Table2:Stability of CBZ in solution.
3.4. Comparison of the methods
The proposed analytical methods were compared using
statistical analysis. The F- test was applied and did not reveal
any significant difference between the experimental values
obtained in the sample analysis by the three methods (Table
4). According to the statistical comparison of results, there is
no significant difference between HPLC-DAD, first orderand second order- derivative spectrophotometric methods.
3.3.3. Accuracy and Precision
The precision of the proposed method was
determined by repeatability (within-day) and intermediate
precision (between-day). The precision study was examined
by analysing three different samples (2.5, 7.5 and 15 g ml-1,
quality control samples) by only one operator. The
repeatability was evaluated by analyzed six time within one
day, whereas intermediation precision was evaluated by
analyzed once daily for six days. The precision of HPLCDAD, first order- and second order- derivative
spectrophotometric methods were demonstrated by RSD % of
lower than 6,68, 9,64, 9.56 %, respectively. These data are
summarised in Table 3
To determine the accuracy of the proposed methods
and to study the interference of formulation additives, the
accuracy was checked as three concentration levels (2, 15, 25
g ml-1, quality control samples) The precision of HPLCDAD, first order- and second order- derivative
spectrophotometric methods were demonstrated by relative
error % of lower than 6,0, 9,86, 11,6 %, respectively. These
data are also summarised in Table 3
Table 3: Precision of the methods for determination of CBZ
2.SDa : Standard deviation of six replicate determinations, RSD: Relative
standard derivation, bAverage of six replicate determinations, Accuracyc :
(relative error%)= (Measured amount - Actual amount )/ Actual amount
x100
3.3.4. Sensitivity
Table 4: Statistical evaluation of obtained data from three
methods in tablets containing CBZ.
n: Number of determination, aX: mean, SD: standart deviation, CI
:Confidence interval Ft: Tabulated F values (Ft : 3.330) Fc: Calculated F
values, Ho: Hypothesis: no statitically significant difference exists among
three methods, Ft Fc; , Ho hypothesis in accepted (P <0.05).
4.CONCLUSION
The first order- and second order- derivative
spectrophotometric and HPLC-DAD methods were
developed and validated successfully in the quantitative
analysis of carbamazepine in pure forms and in
pharmaceutical preparations. The most striking features of
derivative spectrophotometric methods described here are
their simplicity and rapidity, no requiring time-consuming
sample preparation such as filtration, degassing that are need
for HPLC procedure and also chromatographic methods need
expensive equipment. The assay results obtained these three
methods are in intended agreement. The described derivative
spectrophotometric methods are an excellent alternative to
HPLC method for determination of the CBZ in tablet or a
corresponding mixture and all the reported methods can be
used for the routine quality control analysis of the
investigated drug in pharmaceutical preparations.
REFERENCES
Auer, M.E., U.S. Griesser, J. Sawatzki, Qualitative and
quantitative study of polymorphic forms in drug
formulations by near infrared FT-Raman spectroscopy.
Journal of Molecular Structure, 661: 307-317.
Camara, M.S., C. Mastandrea, H.C. Goicoechea (2005).
Chemometrics-assisted
simple
UV-spectroscopic
determination of carbamazepine in human serum and
comparision with reference methods. J.Biochem
Biophys.Methods, 64: 153-166.
Chen, K., K.K. Bashi (1991) Comperative analysis of
antiepileptic drugs by gas chromatography using capillary
or packed columns and by fluorescens polarization
immunoassay, Analytical Toxicol, 15: 82-85.
Fellenberg, A.J., and A.C. Pollard (1976). A rapid
spectrophotometric procedure for the simultaneous micro
determination of carbamazepine and 5,5-diphenylhydantoin in blood. Clinica Chimica Acta, 69: 429-431.
Frigerio, A., K.M. Baker, G. Belvedere (1973). Gas
chromatographic degradation of several drugs and their
metabolites.Analytical Chemistry, 45: 1846-1850.
González-Barreiro, C., M. Lores, M.C. Casais, R. Cela
(2003). Simultaneous determination of neutral and acidic
pharmaceuticals in wastewater by high-performance
liquid chromatography–post-column photochemically
induced fluorimetry, Journal of Chromatography A, 993:
29-37.
Huang, C., Q. He, H. Chen (2002). Flow injection
photochemical spectrofluorimetry for the determination of
carbamazepine in pharmaceutical preparations. Journal of
Pharmaceutical and Biomedical Analysis, 22: 59-65
Jung, H. R.C. Milan, M.E. Girard, F. Leon, M.A. Montoya
(1997). Bioequivalence study of carbamazepine tablets: in
vitro/in vivo correlation. Int. J. of Pharmaceutics,152:
37-44.
Lam, M.W., C.J. Young, R.A. Brain, D.J. Johnson, M.A.
Hanson, C.J. Wilson, S.M. Richards, K.R. Solamon, S.A.
Mabury (2004). Aquatic persistence of eight
pharmaceutical in a microcom study. Toxicology and
Chemistry, 23: 1431-1440.
Manoj Babu, M.K. (2004). Simultaneous separation and
quantitation of four antiepileptic drugs a study with
potential for use in patient drug level monitoring. Journal
of Pharmaceutical and Biomedical Analysis, 34: 315-324.
Mohammed, E.A.H.(2000). Comparative LC–MS and HPLC
analyses of selected antiepileptics and beta-blocking
drugs. Il Farmaco, 55: 136-145.
Olling, M., T.T. Mensinga, D.M. Barends, C. Groen, O.A.
Lake, J. Meulenbent (1999). Bioavailability of
carbamazepine from four different products and the
occurrence of side effects. Biopharmmaceutics and Drug
Disposition, 20:19-28.
Rezaei, Z., B. Hemmateenejad, S. Khabnadideh, M. Gorgin
(2005). Simultaneous spectrophotometric determination
of carbamazepine and phenytoin in serum by PLS
regression and comparison with HPLC. Talanta, 65: 2128.
Riad, L.E., K.K. Chan, W.E. Wagner, R.J. Sawchuk (1986).
Simultaneous first and zero-order absorption of
carbamazepine tablets in humans. Journal of
Pharmacological Sciences, 75: 897-900.
Validation of Analytical Procedures, Proceedings of the
International Conference on Harmonisation (ICH),
Commision of the European Communities, 1996.